The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study is protein kinases.
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (See, Hardie, G. and Hanks, S. The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576-596; Knighton et al., Science 1991, 253, 407-414; Hiles et al., Cell 1992, 70, 419-429; Kunz et al., Cell 1993, 73, 585-596; Garcia-Bustos et al., EMBO J. 1994, 13, 2352-2361).
In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease, and hormone-related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.
The Janus kinases (JAK) are a family of tyrosine kinases consisting of JAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokine signaling. The down-stream substrates of the JAK family of kinases include the signal transducer and activator of transcription (STAT) proteins. JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis as well as in solid and hematologic malignancies such as leukemias and lymphomas. The pharmaceutical intervention in the JAK/STAT pathway has been reviewed [Frank Mol. Med. 5, 432-456 (1999) & Seidel, et al, Oncogene 19, 2645-2656 (2000)].
JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 is predominantly expressed in hematopoietic cells. JAK3 binds exclusively to the common cytokine receptor gamma chain (γc) and is activated by IL-2, IL-4, IL-7, IL-9, and IL-15. The proliferation and survival of murine mast cells induced by IL-4 and IL-9 have, in fact, been shown to be dependent on JAK3- and γc-signaling [Suzuki et al, Blood 96, 2172-2180 (2000)].
Cross-linking of the high-affinity immunoglobulin (Ig) E receptors of sensitized mast cells leads to a release of proinflammatory mediators, including a number of vasoactive cytokines resulting in acute allergic, or immediate (type I) hypersensitivity reactions [Gordon et al, Nature 346, 274-276 (1990) & Galli, N. Engl. J. Med., 328, 257-265 (1993)]. A crucial role for JAK3 in IgE receptor-mediated mast cell responses in vitro and in vivo has been established [Malaviya, et al, Biochem. Biophys. Res. Commun. 257, 807-813 (1999)]. In addition, the prevention of type I hypersensitivity reactions, including anaphylaxis, mediated by mast cell-activation through inhibition of JAK3 has also been reported [Malaviya et al, J. Biol. Chem. 274, 27028-27038 (1999)]. Targeting mast cells with JAK3 inhibitors modulated mast cell degranulation in vitro and prevented IgE receptor/antigen-mediated anaphylactic reactions in vivo.
A recent study described the successful targeting of JAK3 for immune suppression and allograft acceptance. The study demonstrated a dose-dependent survival of Buffalo heart allograft in Wistar Furth recipients upon administration of inhibitors of JAK3 indicating the possibility of regulating unwanted immune responses in graft versus host disease [Kirken, Transpl. Proc. 33, 3268-3270 (2001)].
IL-4-mediated STAT-phosphorylation has been implicated as the mechanism involved in early and late stages of rheumatoid arthritis (RA). Up-regulation of proinflammatory cytokines in RA synovium and synovial fluid is a characteristic of the disease. It has been demonstrated that IL-4-mediated activation of IL-4/STAT pathway is mediated through the Janus Kinases (JAK 1 & 3) and that IL-4-associated JAK kinases are expressed in the RA synovium [Muller-Ladner, et al, J. Immunol. 164, 3894-3901 (2000)].
Familial amyotrophic lateral sclerosis (FALS) is a fatal neurodegenerative disorder affecting about 10% of ALS patients. The survival rates of FALS mice were increased upon treatment with a JAK3 specific inhibitor. This suggested that JAK3 plays a role in FALS [Trieu, et al, Biochem. Biophys. Res. Commun. 267, 22-25 (2000)].
Signal transducer and activator of transcription (STAT) proteins are activated by, among others, the JAK family kinases. Results form a recent study suggested the possibility of intervention in the JAK/STAT signaling pathway by targeting JAK family kinases with specific inhibitors for the treatment of leukemia [Sudbeck, et al, Clin. Cancer Res. 5, 1569-1582 (1999)]. JAK3 specific compounds were shown to inhibit the clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LC1; 19, NALM-6, MOLT-3 and HL-60.
In animal models, TEL/JAK2 fusion proteins have induced myeloproliferative disorders and in hematopoietic cell lines, introduction of TEL/JAK2 resulted in activation of STAT 1, STAT3, STAT5, and cytokine-independent growth [Schwaller, et al, EMBO J. 17, 5321-5333 (1998)].
Inhibition of JAK 3 and TYK 2 abrogated tyrosine phosphorylation of STAT3, and inhibited cell growth of mycosis fungoides, a form of cutaneous T cell lymphoma. These results implicated JAK family kinases in the constitutively activated JAK/STAT pathway that is present in mycosis fungoides [Nielsen, et al, Proc. Nat. Acad. Sci. U.S.A. 94, 6764-6769 (1997)]. Similarly, STAT3, STAT5, JAK1 and JAK2 were demonstrated to be constitutively activated in mouse T cell lymphoma characterized initially by LCK over-expression, thus further implicating the JAK/STAT pathway in abnormal cell growth [Yu, et al, J. Immunol. 159, 5206-5210 (1997)]. In addition, IL-6-mediated STAT3 activation was blocked by an inhibitor of JAK, leading to sensitization of myeloma cells to apoptosis [Catlett-Falcone, et al, Immunity 10, 105-115 (1999)].
One kinase family of interest is Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK), which is believed to be an effector of Ras-related small GTPase Rho. The ROCK family includes p160ROCK (ROCK-1) (Ishizaki et al., EMBO J. 1996, 15, 1885-1893) and ROKα/Rho-kinase/ROCK-II (Leung et al., J. Biol. Chem. 1995, 270, 29051-29054; Matsui et al., EMBO J. 1996, 15, 2208-2216; Nakagawa et al., FEBS Lett. 1996, 392, 189-193), protein kinase PKN (Amano et al., Science 1996, 271, 648-650; Watanabe et al., Science 1996, 271, 645-648), and citron and citron kinase (Madaule et al. Nature, 1998, 394, 491-494; Madaule et al., FEBS Lett. 1995, 377, 243-248). The ROCK family of kinases have been shown to be involved in a variety of functions including Rho-induced formation of actin stress fibers and focal adhesions (Leung et al., Mol. Cell. Biol. 1996, 16, 5313-5327; Amano et al., Science, 1997, 275, 1308-1311; Ishizaki et al., FEBS Lett. 1997, 404, 118-124) and in downregulation of myosin phosphatase (Kimura et al., Science, 1996, 273, 245-248), platelet activation (Klages et al., J. Cell. Biol., 1999, 144, 745-754), aortic smooth muscle contraction by various stimuli (Fu et al., FEBS Lett., 1998, 440, 183-187), thrombin-induced responses of aortic smooth muscle cells (Seasholtz et al., Cir. Res., 1999, 84, 1186-1193), hypertrophy of cardiomyocytes (Kuwahara et al., FEBS Lett., 1999, 452, 314-318), bronchial smooth muscle contraction (Yoshii et al, Am. J. Respir. Cell Mol. Biol., 1999, 20, 1190-1200), smooth muscle contraction and cytoskeletal reorganization of non-muscle cells (Fukata et al, Trends in Pharm. Sci 2001, 22, 32-39), activation of volume-regulated anion channels (Nilius et al., J. Physiol., 1999, 516, 67-74), neurite retraction (Hirose et al., J. Cell. Biol., 1998, 141, 1625-1636), neutrophil chemotaxis (Niggli, FEBS Lett., 1999, 445, 69-72), wound healing (Nobes and Hall, J. Cell. Biol., 1999, 144, 1235-1244), tumor invasion (Itoh et al, Nat. Med., 1999, 5, 221-225) and cell transformation (Sahai et al., Curr. Biol., 1999, 9, 136-145).
More specifically, ROCK has been implicated in various diseases and disorders including hypertension (Satoh et al., J. Clin. Invest. 1994, 94, 1397-1403; Mukai et al., FASEB J. 2001, 15, 1062-1064; Uehata et al, Nature 1997, 389, 990-994; Masumoto et al., Hypertension, 2001, 38, 1307-1310), cerebral vasospasm (Sato et al, Circ. Res. 2000, 87, 195-200; Miyagi et al., J. Neurosurg. 2000, 93, 471-476; Tachibana et al., Acta Neurochir (Wien) 1999, 141, 13-19), coronary vasospasm (Shimokawa et al., Jpn. Cir. J. 2000, 64, 1-12; Kandabashi et al., Circulation 2000, 101, 1319-1323; Katsumata et al., Circulation 1997, 96, 4357-4363; Shimokawa et al., Cardiovasc. Res. 2001, 51, 169-177; Utsunomiya et al., J. Pharmacol. 2001, 134, 1724-1730; Masumoto et al., Circulation 2002, 105, 1545-1547), bronchial asthma (Chiba et al., Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 1995, 11, 351-357; Chiba et al., Br. J. Pharmacol. 1999, 127, 597-600; Chiba et al., Br. J. Pharmacol. 2001, 133, 886-890; Tizuka et al., Eur. J. Pharmacol. 2000, 406, 273-279), preterm labor (Niro et al., Biochem. Biophys. Res. Commun. 1997, 230, 356-359; Tahara et al., Endocrinology 2002, 143, 920-929; Kupittayanant et al., Pflugers Arch. 2001, 443, 112-114), erectile dysfunction (Chitaley et al., Nat. Med. 2001, 7, 119-122; Mills et al., J. Appl. Physiol. 2001, 91, 1269-1273), glaucoma (Honjo et al., Arch. Opthalmol. 2001, 1171-1178; Rao et al., Invest. Opthalmol. Vis. Sci. 2001, 42, 1029-1037), vascular smooth muscle cell proliferation (Shimokawa et al., Cardiovasc. Res. 2001, 51, 169-177; Morishige et al, Arterioscler. Thromb. Vasc. Biol. 2001, 21, 548-554; Eto et al., Am. J. Physiol. Heart Circ. Physiol. 2000, 278, H1744-H1750; Sawada et al., Circulation 2000, 101, 2030-2023; Shibata et al., Circulation 2001, 103, 284-289), myocardial hypertrophy (Hoshijima et al., J. Biol. Chem. 1998, 273, 7725-77230; Sah et al., J. Biol. Chem. 1996, 271, 31185-31190; Kuwahara et al., FEBS Lett. 1999, 452, 314-318; Yanazume et al., J. Biol. Chem. 2002, 277, 8618-8625), malignoma (Itoh et al., Nat. Med. 1999, 5, 221-225; Genda et al., Hepatology 1999, 30, 1027-1036; Somlyo et al., Biochem. Biophys. Res. Commun. 2000, 269, 652-659), ischemia/reperfusion-induced injury (Ikeda et al., J. of Surgical Res. 2003, 109, 155-160; Miznuma et al. Transplantation 2003, 75, 579-586), endothelial dysfunction (Hernandez-Perera et al, Circ. Res. 2000, 87, 616-622; Laufs et al., J. Biol. Chem. 1998, 273, 24266-24271; Eto et al., Circ. Res. 2001, 89, 583-590), Crohn's Disease and colitis (Segain et al. Gastroenterology 2003, 124(5), 1180-1187), neurite outgrowth (Fournier et al. J. Neurosci. 2003, 23, 1416-1423), Raynaud's Disease (Shimokawa et al. J. Cardiovasc. Pharmacol. 2002, 39, 319-327), angina (Utsunomiya et al. Br. J. Pharmacol. 2001, 134, 1724-1730; Masumoto et al, Circulation 2002, 105, 1545-1547; Shimokawa et al, J. Cardiovasc. Pharmacol., 2002, 40, 751-761; Satoh et al., Jpn. J. Pharmacol., 2001, 87, 34-40), Alzheimer's disease (Zhou et al., Science 2003, 302, 1215-1218), benign prostatic hyperplasia (Rees et al., J. Urology, 2003, 170, 2517-2522), and atherosclerosis (Retzer et al. FEBS Lett. 2000, 466, 70-74; Ishibashi et al. Biochim. Biophys. Acta 2002, 1590, 123-130). Accordingly, the development of inhibitors of ROCK kinase would be useful as therapeutic agents for the treatment of disorders implicated in the ROCK kinase pathway.
The Aurora proteins are a family of three highly related serine/threonine kinases (termed Aurora-A, -B and -C) that are essential for progression through the mitotic phase of cell cycle. Specifically Aurora-A plays a crucial role in centrosome maturation and segregation, formation of the mitotic spindle and faithful segregation of chromosomes. Aurora-B is a chromosomal passenger protein that plays a central role in regulating the alignment of chromosomes on the meta-phase plate, the spindle assembly checkpoint and for the correct completion of cytokinesis.
Overexpression of Aurora-A, -B or -C has been observed in a range of human cancers including colorectal, ovarian, gastric and invasive duct adenocarcinomas. In addition amplification of the AURKA locus that encodes for Aurora-A correlates with poor prognosis for patients with node-negative breast cancer. Furthermore overexpression of Aurora-A has been shown to transform mammalian fibroblasts, giving rise to aneuploid cells containing multipolar spindles.
A number of studies have now demonstrated that depletion or inhibition of Aurora-A or -B in human cancer cell lines by siRNA, dominant negative or neutralising antibodies disrupts progression through mitosis with accumulation of cells with 4N DNA, and in some cases this is followed by endoreduplication and cell death.
Protein kinases are attractive and proven targets for new therapeutic agents to treat a range if human diseases, with examples including Gleevec and Tarceva. The Aurora kinases are especially attractive due to their association with numerous human cancers and the role they play in promoting proliferation of these cancer cells (Harrington et al., Nature Med., 2004, 10: 262-267).
Accordingly, there is a great need to develop inhibitors of JAK, ROCK and Aurora, preferably JAK-3, ROCK and Aurora A, protein kinases that are useful in treating various diseases or conditions associated with JAK, ROCK and Aurora activation, particularly given the inadequate treatments currently available for the majority of these disorders.